Bilirubin Acts as a Direct Agonist of TRPM2 Channel to Exacerbate Ischemic Brain Injury: Insights Empowered by ANT BIO PTE. LTD.

Stroke, commonly referred to as cerebrovascular accident, is one of the leading causes of adult disability and mortality worldwide. It is primarily triggered by the interruption of cerebral blood perfusion due to conditions such as atherosclerosis, atrial fibrillation, hypertension, and diabetes. Stroke is categorized into ischemic and hemorrhagic subtypes, with ischemic stroke accounting for approximately 80% of all cases. During ischemic stroke, the massive release of glutamate activates N-methyl-D-aspartate receptors (NMDARs), inducing calcium influx into neurons through these ion channels, which leads to neuronal calcium overload and disruption of calcium homeostasis. Despite extensive research on ischemic brain injury, effective therapeutic agents remain scarce. Therefore, there is an urgent need to elucidate the underlying mechanisms of stroke and identify potential novel targets for the treatment of ischemic stroke.

On March 14, 2023, the teams led by Shankai Yin, Haibo Shi from Shanghai Jiao Tong University, and Lu-Yang Wang from the University of Toronto published a research paper entitled "Bilirubin gates the TRPM2 channel as a direct agonist to exacerbate ischemic brain damage" online in Neuron. The study proposed that bilirubin, as an endogenous agonist, can directly activate the TRPM2 channel, thereby exacerbating brain damage in ischemic stroke. Through animal experiments, the research team demonstrated that molecular intervention and TRPM2 channel blockade in mouse models exert significant neuroprotective effects. Notably, multiplex fluorescence immunohistochemistry (mIHC) kits from the Absin product line of ANT BIO PTE. LTD. played a crucial role in validating the expression and localization of key molecules (e.g., TRPM2, NeuN, BVR, HO-1) in neurons during stroke, providing direct evidence for the proposed mechanism.

1. Literature Information

•         Title: Bilirubin gates the TRPM2 channel as a direct agonist to exacerbate ischemic brain damage

•         Journal: Neuron

•         Publication Date: Online on March 14, 2023; Published on May 17, 2023

•         DOI: 10.1016/j.neuron.2023.02.022

•         Research Team: Shankai Yin, Haibo Shi et al., Shanghai Jiao Tong University; Lu-Yang Wang et al., University of Toronto

•         PMID: 36921602

•         PMCID: PMC10191619

•         Core Reagents from ANT BIO PTE. LTD.: Multiplex Fluorescence IHC Staining Kits (Absin product line), including 4-color to 7-color plus kits (e.g., Catalog No.: abs50012, abs50015) and supporting reagent Antibody Elution Buffer (mIHC-Specific, Catalog No.: abs994)

•         Related Product Link: AntBio - Fueling Research, Feeding Discovery

2. Research Background

The role of bilirubin in stroke remains controversial. Previous studies have shown that patients with high serum bilirubin levels exhibit more severe neurological symptoms, but the specific mechanism by which bilirubin affects ischemic brain injury is unclear. Under ischemic conditions, increased intracellular Ca²⁺ concentration induces excessive production of reactive oxygen species (ROS), which activates downstream signaling pathways and leads to cell death. TRPM2, a member of the transient receptor potential melastatin (TRPM) family, is a Ca²⁺-permeable ion channel that is activated by ADPR and ROS, and has been implicated in various neurological diseases. However, whether bilirubin interacts with TRPM2 and the nature of this interaction in the context of ischemic brain injury have not been previously reported. Clarifying this interaction could provide novel insights into the pathogenesis of ischemic stroke and identify new therapeutic targets.

3. Research Strategy

The research team employed a comprehensive, multi-disciplinary approach to systematically explore the role and mechanism of bilirubin-TRPM2 interaction in ischemic brain injury:

1.       Clinical Correlation Analysis: Analyzed clinical records of stroke patients to investigate the relationship between serum total bilirubin (TB) and direct bilirubin (DB) levels and infarct volume, with a focus on patients with hepatitis B.

2.       Animal Model Experiments: Utilized transient middle cerebral artery occlusion (tMCAO) animal models and Trpm2 genotype mice (Trpm2⁺/⁺ and Trpm2⁻/⁻) to evaluate the effect of bilirubin on infarct volume. Measured bilirubin levels in serum and cerebrospinal fluid (CSF) and their correlation with infarct volume.

3.       Mechanistic Exploration of Bilirubin-TRPM2 Interaction: Introduced point mutations in the ADPR-binding sites and NUDT9-H domain of TRPM2 to investigate the activation mechanism of bilirubin on TRPM2. Performed truncation experiments of N-terminal and C-terminal ADPR-binding domains. Used molecular docking simulations and molecular dynamics simulations to identify the binding pocket of bilirubin on TRPM2 and compare the binding affinities of bilirubin and XAME.

4.       Validation of Bilirubin Binding Pocket: Substituted key amino acid residues (D866, K928, D1069) in the predicted bilirubin binding pocket with non-polar alanine to verify their role in bilirubin-induced TRPM2 activation. Used electrophysiological experiments to assess the effect of these mutations on channel activity.

5.       Spatial Localization and Expression Validation: Employed multiplex fluorescence IHC (using ANT BIO PTE. LTD.’s Absin kits) to detect the co-expression and localization of NeuN (neuronal marker), TRPM2, biliverdin reductase (BVR), and heme oxygenase 1 (HO-1) in brain tissues of stroke models, confirming the production and release of bilirubin in neurons.

6.       Therapeutic Potential Evaluation: Investigated the effect of TRPM2 blockers (e.g., clotrimazole, talM2NX, A23) on bilirubin-induced neurotoxicity. Used gene knock-in mice (D1066A) to verify the role of the bilirubin binding pocket in neurotoxicity. Evaluated the neuroprotective effect of A23 in tMCAO models combined with bilirubin injection.

4. Key Research Findings

4.1 Bilirubin Levels Correlate Positively with Infarct Volume in Stroke Patients and Mouse Models

Clinical data analysis showed that serum TB and DB levels in stroke patients with hepatitis B were 2 to 3 times higher than those in the normal group. Stroke significantly increased TB and DB concentrations, indicating that elevated bilirubin levels may be a result of stroke and are positively correlated with infarct volume. Animal experiments demonstrated that bilirubin injection increased infarct volume in Trpm2⁺/⁺ mice with tMCAO, but had no effect on infarct volume in Trpm2⁻/⁻ mice. Bilirubin levels in serum and CSF were significantly correlated with infarct volume. These results suggest that ischemia-reperfusion during stroke leads to increased bilirubin concentration, and TRPM2 channels play a regulatory role in this process.

4.2 Bilirubin Activates TRPM2 Channels Through a Novel Gating Mechanism

Mutations in ADPR-binding sites and NUDT9-H domain of TRPM2 did not abolish the response to bilirubin, although the activation amplitude was reduced, whereas ADPR failed to activate the mutated TRPM2 channels. Truncation of N-terminal and C-terminal ADPR-binding domains blocked ADPR-induced TRPM2 activation but did not affect bilirubin-induced activation. Molecular docking simulations revealed that bilirubin may interact with a specific binding pocket on TRPM2, located near the calcium-binding site, forming hydrogen bonds and salt bridges with amino acid residues such as D866, K928, and D1069. Substitution of these residues with alanine completely blocked bilirubin-induced activation but did not affect ADPR and calcium-induced activation. Molecular dynamics simulations showed that XAME had higher affinity for TRPM2 than bilirubin, which may explain the differences in their activation speed and efficacy. These findings reveal a unique mechanism by which bilirubin activates TRPM2 channels, distinct from classical intracellular ligands.

4.3 The Bilirubin-Binding Cavity in TRPM2 is an Ideal Drug Target for Antagonizing Neurotoxicity

TRPM2 is a potential therapeutic target for stroke. Previous preclinical studies have shown that TRPM2 blockers such as clotrimazole, talM2NX, and A23 have neuroprotective effects. The study found that the newly reported TRPM2 blocker A23 forms a highly stable binding with the bilirubin-binding cavity, providing a structural explanation for its in vitro nanomolar half-maximal inhibitory concentration (IC₅₀) and in vivo efficacy against ischemic injury. A23 has high affinity for the bilirubin-binding cavity and can effectively antagonize bilirubin-induced neurotoxicity. Multiplex IHC results confirmed the co-expression of NeuN, TRPM2, BVR, and HO-1 in neurons, indicating that bilirubin is mainly produced and released in neurons during stroke. Ischemia and hypoxia can directly induce the release of endogenous bilirubin in the brain, thereby exacerbating neurotoxicity during stroke.

4.4 Molecular Disruption of the Bilirubin-Binding Cavity in TRPM2 Eliminates Bilirubin-Induced Excitability Upregulation and Neurotoxicity

Bilirubin and its structurally similar derivatives activate TRPM2 channels by binding to the cavity near the channel, and A23 blocks the binding of these agonists through competitive antagonism. To verify that the cavity on TRPM2 mediates bilirubin neurotoxicity, the research team used a gene knock-in mouse line in which key residues of TRPM2 were replaced with alanine (D1066A). Electrophysiological experiments showed that bilirubin no longer induced excessive neuronal excitation in D1066A mice, and the exacerbating effect of bilirubin on ischemic brain injury was completely inhibited in the ischemic brain injury model. This indicates that the binding cavity in TRPM2 plays a key role in bilirubin-induced neurotoxicity in stroke.

5. Product Empowerment: The Critical Role of ANT BIO PTE. LTD.’s Multiplex IHC Kits

Validating the production and release site of bilirubin in neurons during stroke was a key step in this study, as it provided direct evidence for the neurotoxic effect of endogenously produced bilirubin. ANT BIO PTE. LTD.’s Absin multiplex fluorescence IHC kits delivered the high-performance multiplexing capability, sensitivity, and specificity required for this critical validation, enabling the simultaneous detection of multiple key molecules in brain tissue sections.

5.1 Core Products and Their Application Value

5.2 Technical Value in Neurological Disease Research

In neurological disease research, the accurate localization and co-expression analysis of key molecules in specific cell types are critical for understanding disease mechanisms. ANT BIO PTE. LTD.’s multiplex fluorescence IHC kits enable researchers to simultaneously detect multiple key molecules on a single tissue section, preserving the native spatial context of molecular expression—a significant advantage over traditional single-marker IHC. In this study on ischemic brain injury, the kits’ high sensitivity and specificity ensured clear detection of low-abundance molecules in complex brain tissue samples, while their ability to multiplex multiple markers allowed for the comprehensive analysis of the co-expression of TRPM2, BVR, HO-1, and neuronal markers. This technical empowerment was instrumental in validating the neuronal origin of bilirubin during stroke, highlighting the value of ANT BIO PTE. LTD.’s products in neurological disease research.

6. Brand Mission

As a professional supplier of life science reagents, ANT BIO PTE. LTD. is dedicated to providing high-quality, reliable products and comprehensive solutions to empower global life science research. The company's three specialized sub-brands cover the full spectrum of research needs in the life science field: Absin focuses on general reagents and kits, Starter specializes in antibodies, and UA is dedicated to recombinant proteins. Our core mission is to bridge the gap between cutting-edge scientific research and practical applications, accelerate the pace of scientific discovery, and contribute to the advancement of human health and regenerative medicine.

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8. Disclaimer

This article is AI-compiled and interpreted based on the original work in DOI: 10.1002/advs.202413562. All intellectual property (e.g., images, data) of the original publication shall belong to the journal and the research team. For any infringement, please contact us promptly and we will take immediate action.

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At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.